Epigenetic changes in preimplantation embryos subjected to laser manipulation.

The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6-8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 μs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.